A. Field of the Invention
The present invention relates to fluorescent structural analogs of the non-fluorescent nucleosides commonly found in DNA and RNA, methods of their derivatization and subsequent use in the synthesis of fluorescent oligonucleotides, and to their new and useful applications both as fluorescent monomers and in fluorescent oligonucleotides having prescribed sequences. Additionally, it relates to applications in which fluorescent structural analogs are substituted for specific non-fluorescent nucleosides in prescribed DNA or RNA sequences and to methods of using fluorescent oligonucleotides as hybridization reagents and probes for diagnostic and therapeutic purposes and as diagnostic and therapeutic research tools.
B. General Description of the Art
The six commonly occurring N-nucleosides which predominate in the composition of DNA and RNA from all sources have the structures shown in FIG. 1 wherein R.sub.6 is H for inosine and NH.sub.2 for guanosine, R.sub.9 is H for uridine and CH.sub.3 for thymidine. Furthermore, R.sub.12, R.sub.14 =OH for ribonucleotides, R.sub.12 =OH, R.sub.14 =H for 2'-deoxy nucleotides, R.sub.12 =H, R.sub.14 =OH for 3'-deoxy nucleotides, and R.sub.12, R.sub.14 =H in dideoxy nucleotides.
The six commonly occurring nucleotides do not absorb light at wavelengths &gt;290 nm and are effectively non-fluorescent under physiological conditions. Derivatives of the commonly occurring N-nucleotides for a variety of synthetic, diagnostic, and therapeutic purposes are common, including substitutions on both the heterocyclic base and the furanose ring. These substitutions can be made at the loci shown in FIG. 2 in which R.sub.4 is a reactive group derivatizible with a detectable label (NH.sub.2, SH, .dbd.O, and which can include an optional linking moiety including, but not limited to, an amide, thioether, or disulfide linkage or a combination thereof with additional variable reactive groups, R.sub.1 through R.sub.3, e.g., R.sub.1 --(CH.sub.2).sub.x --R.sub.2, or R.sub.1 -R.sub.2 --(CH.sub.2).sub.x --R.sub.3 --, where x is an integer in the range of 1 and 25 inclusive; and R.sub.1, R.sub.2, and R.sub.3 can be H, OH, alkyl, acyl, amide, thioether, or disulfide); R.sub.5 is H or part of an etheno linkage with R.sub.4 ; R.sub.6 is H, NH.sub.2, SH, or .dbd.O; R.sub.9 is hydrogen, methyl, bromine, fluorine, or iodine, or an alkyl or aromatic substituent, or an optional linking moiety including an amide, thioether, or disulfide linkage or a combination thereof such as R.sub.1 --(CH.sub.2).sub.x --R.sub.2, or R.sub.1 -R.sub.2 --(CH.sub.2).sub.x --R.sub.3 --, where x is an integer in the range of 1 and 25 inclusive; R.sub.10 is hydrogen, or an acid-sensitive base stable blocking group, or a phosphorous derivative, R.sub.11 =R.sub.12 =H; R.sub.12 is hydrogen, OH, or a phosphorous derivative; R.sub.14 is H, OH, or OR.sub.3 where R.sub.3 is a protecting group or additional fluorophore. The letters N and C in the N-nucleosides and C-nucleosides designate the atom at which the glycosidic covalent bond connects the sugar and the heterocyclic base. In the cases of the commonly occurring nucleosides, the bases are either adenine, guanine, cytosine, inosine, uracil, or thymine. The bases are attached to a furanose sugar, a general structure of which is shown in FIG. 3. The sugar substituents for the fluorescent analogs share the same numbering system for all R groups, but the numbering system for some of the heterocycle analogs may differ.
I. Known Methods of Labeling Nucleotides
Nucleotide sequences are commonly utilized in a variety of applications including diagnostic and therapeutic probes which hybridize target DNA and RNA and amplification of target sequences. It is often necessary, or useful, to label nucleotide sequences.
A. Labeling of oligonucleotide probes with radioisotopes. Hybridization of specific DNA or RNA sequences typically involves annealing oligonucleotides of lengths which range from as little as 5 bases to more than 10,000 bases (10 kb). The majority of oligonucleotide probes currently in research use are radioactively labeled; however, because of (a) the short half lives of the isotopes in common usage, (b) the safety requirements, and (c) the costs of handling and disposal of radioactive probes, convenient and sensitive non-isotopic methods of detection are required for hybridization diagnostic methods to achieve widespread acceptance and application.
B. Non-isotopic methods of labeling oligonucleotide probes. In general, all of the non-isotopic methods of detecting hybridization probes that are currently available depend on some type of derivatization of the nucleotides to allow for detection, whether through antibody binding, or enzymatic processing, or through the fluorescence or chemiluminescence of an attached "reporter" molecule. In most cases, oligonucleotides have been derivatized to incorporate single or multiple molecules of the same reporter group, generally at specific cyclic or exocyclic positions. Techniques for attaching reporter groups have largely relied upon (a) functionalization of 5' or 3' termini of either the monomeric nucleosides or the oligonucleotide strands by numerous chemical reactions using deprotected oligonucleotides in aqueous or largely aqueous media (see Cardullo et al. [1988] PNAS 85:8790-8794); (b) synthesizing modified nucleosides containing (i) protected reactive groups, such as NH.sub.2, SH, CHO, or COOH, (ii) activatable monofunctional linkers, such as NHS esters, aldehydes, or hydrazides, or (iii) affinity binding groups, such as biotin, attached to either the heterocyclic base or the furanose moiety. Modifications have been made on intact oligonucleotides or to monomeric nucleosides which have subsequently been incorporated into oligonucleotides during chemical synthesis via terminal transferase or "nick translation" (see, e.g., Brumbaugh et al. [1988] PNAS 85:5610-5614; Sproat, B. S., A. I. Lamond, B. Beijer, P. Neuner, P. Ryder [1989] Nucl. Acids Res. 17:3371-3386; Allen, D. J., P. L. Darke, S. J. Benkovic [1989] Biochemistry 28:4601-4607); (c) use of suitably protected chemical moieties, which can be coupled at the 5' terminus of protected oligonucleotides during chemical synthesis, e.g., 5'-aminohexyl-3'-O-phosphoramidite (Haralambidis, J., L. Duncan, G. W. Tregar [1990] Nucl. Acids Res. 18:493-499); and, (d) addition of functional groups on the sugar moiety or in the phosphodiester backbone of the polymer (see Conway, N. E., J. Fidanza, L. W. McLaughlin [1989] Nucl. Acids Res. Symposium Series 21:43-44; Agrawal, S., P. C. Zamecnik [1990] Nucl. Acids Res. 18:5419-5423).
At the simplest, non-nucleoside linkers and labels have been attached to the 3' or 5' end of existing oligonucleotides by either enzymatic or chemical methods. Modification of nucleoside residues internal to the sequence of a DNA or RNA strand has proven to be a difficult procedure, since the reaction conditions must be mild enough to leave the RNA or DNA oligomers intact and still yield reaction products which can participate in normal Watson-Crick base pairing and stacking interactions (see FIG. 4).
C. Derivatizations of the heterocyclic base (B). Numerous methods for both cyclic and exocyclic derivatization of the N-nucleoside base have been described, including the following:
(1) Hapten labeling. DNA probes have been amino modified and subsequently derivatized to carry a hapten such as 2,4-dinitrophenol (DNP) to which enzyme-conjugated anti-hapten antibodies bind which subsequently can be processed using a colorimetric substrate as a label (Keller et al. [1988] Analytical Biochemistry 170:441-450). PA1 (2) Amino- and thiol-derivatized oligonucleotides. Takeda and Ikeda ([1984] Nucl. Acids Research Symposium Series 15:101-104) used phosphotriester derivatives of putresceinyl thymidine for the preparation of amino-derived oligomers. Ruth and colleagues have described methods for synthesizing a deoxyuridine analog with a primary amine "linker arm" 12 carbons in length at C.sub.5 (Jablonski et al. [1986] Nucl. Acids Res. 14:6115-6128). These were later reacted with fluorescein to produce a fluorescent molecule. Urdea and Horn were granted a patent in 1990 (U.S. Pat. No. 4,910,300) covering pyrimidine derivatives on which the 6-amino group at C.sub.4 had been modified. 3' and 5' amino modifying phosphoramidites have been widely used in chemical synthesis or derivatized oligonucleotides and are commercially available. PA1 (3) Labeling with photobiotin and other biotinylating agents. The high affinity of biotin for avidin has been used to bind enzymatic or chemiluminescent reagents to derivatized DNA probes (Foster et al. [1985] Nucl. Acids Res. 13:745-761). Biotin conjugated to other linkers has also been widely used, including biotin-NHS esters (Bayer, E. A., M. Wilchek [1980] Methods in Biochemical Analysis 26:1), biotin succinamides (Lee, W. T., D. H. Conrad [1984] J. Exp. Med. 159:1790), and biotin maleimides (Bayer, E. A. et al. [1985] Anal. Biochem. 149:529). Reisfeld et al. ([1987] BBRC 142:519-526) used biotin hydrazide to label the 4-amino group of cytidine. A patent was grained to Klevan et al. in 1989 (U.S. Pat. No. 4,828,979) for such derivatizations at the 6-position of adenfine, the 4-position of cytosine, and the 2-position of guanine. These derivatizations interfere with hydrogen bonding and base-pairing and have limited uses in producing oligomers for use in hybridization. PA1 (4) dU-Biotin labeling. Nucleoside 5'-triphosphates or 3'-O-phosphoramidites were modified with a biotin moiety conjugated to an aliphatic amino group at the 5-position of uracil (Langer et al. [1981] PNAS 78:6633-6637; Saiki et al. [1985] Science 230:1350-1354). The nucleotide triphosphate derivatives are effectively incorporated into double stranded DNA by standard techniques of "nick translation." Once in an oligonucleotide, the residue may be bound by avidin, streptavidin, or anti-biotin antibody which can then be used for detection by fluorescence, chemiluminescence, or enzymatic processing. PA1 (5) 11-digoxigenin-ddUTP labeling. The enzyme, terminal transferase, has been used to add a single digoxigenin-11-dideoxyUTP to the 3' end of oligonucleotides. Following hybridization to target nucleic acids, DIG-ddUTP labeled hybridization probes were detected using anti-DIG antibody conjugate. PA1 (6) AAIF. Immunofluorescent detection can be done using monoclonal Fab' fragments which are specific for RNA:DNA hybrids in which the probe has been derivatized with, e.g., biotin-11-UTP (Bobo et al. [1990] J. Clin. Microbial. 28:1968-1973; Viscidi et al. [1986] J. Clin. Microbiol. 23:311-317). PA1 (7) Bisulfite modification of cytosine. Draper and Gold ([1980] Biochemistry 19:1774-1781) introduced aliphatic amino groups onto cytidine by a bisulfite catalyzed termination reaction; the amino groups were subsequently labeled with a fluorescent tag. In this procedure, the amino group is attached directly to the pyrimidine base. Like the derivatization of uracil, these derivatizations interfere with hydrogen bonding and base-pairing and are not necessarily useful for producing efficient hybridization oligomers. PA1 (8) Fluorophore derivatized DNA probes. Texas Red (Sulfochloro-Rhodamine) derivatized probes are commercially available which hybridize to specific target DNAs and which can be detected using a flow cytometer or a microscope. Numerous authors have reported coupling fluorophores to chemically synthesized oligonucleotides which carried a 5' or 3' terminal amino or thiol group (Brumbaugh et al. [1988] Nucleic Acids Res. 16:4937-4956). PA1 (9) Direct enzyme labeling. Chemical coupling of an enzyme directly to a chemically synthesized probe has been used for direct detection through substrate processing. For example, Urdea et al. described an oligonucleotide sandwich assay in which multiple DNA probe hybridizations were used to bind target DNA to a solid phase after which it was further labeled with additional, alkaline phosphatase-derivatized hybridization probes (Urdea et al. [1989] Clin. Chem. 35:1571-1575). PA1 (10) Acridinium ester labeling. A single phenyl ester of methyl acridinium is attached at a central position on an RNA or DNA probe. Hydrolysis of the ester releases an acridone, CO.sub.2, and light. Because the ester on unhybridized probes hydrolyzes more quickly than the ester on probes which have hybridized to target RNA or DNA, the chemiluminescence of the hybridized probes can be distinguished from that of free probes and is used in a "hybridization protection assay" (Weeks et al. [1983] Clin. Chem. 29:1474-1479). PA1 (1) Internucleotide linkage reporter groups (R.sub.10 site). Phosphorothioate esters have been used to provide a binding site for fluorophores such as monobromobimane (Conway et al. [1989] Nucl. Acids Res. Symposium Series 21:43-44). Agrawal and Zamecnik ([1990] Nucl. Acids Res. 18:5419-5423) reported methods for incorporating amine specific reporter groups (e.g., monobromobimane) and thiol specific reporter groups (e.g., fluorescein isothiocyanate) through modifying the phosphodiester backbone of DNA to phosphoramidites and phosphorothioate diesters, respectively. PA1 (2) Glycosidic reporter groups (R.sub.11 through R.sub.14 sites). Smith, Fung, and Kaiser ([1989] U.S. Pat. No. 4,849,513) described syntheses for an assortment of derivatives and labels on the glycosidic moiety of nucleosides and nucleoside analogs through the introduction of an aliphatic amino group at R.sub.10. The authors did not report or claim any uses or applications of inherently fluorescent oligonucleotides, either made chemically or enzymatically or using the fluorescent nucleoside analogs or their derivatives. PA1 (1) Chemically derivatized dNTPs are generally not cost-effective for use as stock deoxynucleotide triphosphates in PCR amplification, hence, labeling of amplified DNA is limited to (i) amplification using previously labeled primers, or (ii) annealing with labeled hybridization probes. Use of the former frequently results in false positives during amplification owing to (i) non-specific annealing of primers to non-target segments of DNA during amplification, or (ii) contamination by amplicons present in the laboratory environment which are residual from previous amplification experiments. Expense and technical difficulties in post-hybridization processing have largely limited the applications of labeled hybridization probes to research. PA1 (2) Base pairing is hindered for many oligomers made with derivatized nucleosides through the introduction of bulky or non-hydrogen bonding bases at inappropriate sites in a sequence. Owing to the inherent background chemiluminescence of many clinical samples, even the acridinium ester probes have failed to achieve their theoretical levels of sensitivity. The requirements for post hybridization processing have remained a limitation to such methods. PA1 (3) It has proven difficult to provide non-radioactively labeled probes which may be inexpensively produced in large quantities. PA1 (4) Chemiluminescent probes are short lived and samples so tested are difficult to quantify or to "reprobe" accurately. PA1 (5) Hybridization in most cases is only inferred, is non-quantitative or only semi-quantitative, and is non-automatable.
D. Derivatizations of the furanose ring (F). Methods for derivatization of the furanose ring (R.sub.11 through R.sub.14 in FIG. 3) and at the phosphodiester backbone of oligonucleotides (R.sub.10 in FIG. 3) have been reported.
E. Limitations of non-isotopic methods for labeling oligonucleotides. In order to create non-radioactive types of detectable oligonucleotides, it has been necessary to chemically modify the nucleosides typically used in DNA and RNA probes, which has made such probe preparation expensive and laborious; in many cases the detection chemistries have also proven cumbersome and expensive to use, which has largely been responsible for their failure to find significant application in clinical laboratories. In their applications to hybridization, other limitations of chemically derivatized probes have also become apparent.
These limitations have hindered applications of DNA and RNA hybridization probes to clinical laboratory testing and therapeutic uses.
F. Fluorescent N-nucleosides and fluorescent structural analogs. Formycin A (generally referred to as Formycin), the prototypical fluorescent nucleoside analog, was originally isolated as an antitumor antibiotic from the culture filtrates of Nocardia interforma (Hori et al. [1966] J. Antibiotics, Ser. A 17:96-99) and its structure identified as 7-amino-3-b-D-ribafuranosyl (1H-pyrazolo-[4,3d]pyrimidine)) (FIGS. 5 and 6). This antibiotic, which has also been isolated from culture broths of Streptomyces lavendulae (Aizawa et al. [1965] Agr. Biol. Chem. 29:375-376), and Streptomyces gummaensis (Japanese Patent No. 10,928, issued in 1967 to Nippon Kayaku Co., Ltd.), is one of numerous microbial C-ribonucleoside analogs of the N-nucleosides commonly found in RNA from all sources. The other naturally-occurring C-ribonucleosides which have been isolated from microorganisms (FIG. 4) include formycin B (Koyama et al. [1966] Tetrahedron Lett. 597-602; Aizawa et al., supra; Umezawa et al. [1965] Antibiotics Ser. A 18:178-181), oxoformycin B (Ishizuka et al. [1968] J. Antibiotics 21:1-4; Sawa et al. [1968] Antibiotics 21:334-339), pseudouridine (Uematsu and Suahdolnik [1972] Biochemistry 11:4669-4674), showdomycin (Darnall et al. [1967] PNAS 57:548-553), pyrazomycin (Sweeny et al. [1973] Cancer Res. 33:2619-2623), and minimycin (Kusakabe et al. [1972] J. Antibiotics 25:44-47). Formycin, formycin B, and oxoformycin B are pyrazolopyrimidine nucleosides and are structural analogs of adenosine, inosine, and hypoxanthine, respectively; a pyrazopyrimidine structural analog of guanosine obtained from natural sources has not been reported in the literature. A thorough review of the biosynthesis of these compounds is available in Ochi et al. (1974) J. Antibiotics xxiv:909-916.
Physical properties of the nucleoside analogs. Because several of the C-nucleosides were known to be active as antibiotic, antiviral, or anti-tumor compounds, their chemical derivatization and physical properties have been extensively studied and compared to the structures and syntheses of the N-nucleosides commonly found in DNA and RNA. In the late 1960s, several structural analogs of the six commonly occurring N-nucleosides were found to be fluorescent under physiological conditions; fluorescence in the analogs results from a molecular rigidity of the heterocycle structure itself; not all the structural analogs of a given type, e.g., the C-nucleosides, are fluorescent, nor is fluorescence an exclusive or inherent property of any particular class of structural analogs. Our subsequent studies have shown that only a few of the pyrazolo and pyrolo pyrimidines and purines are fluorescent, and that the property is shared with a few other nucleoside derivatives and structural analogs including, but not limited to, several substituted N-nucleosides, azanucleosides, ethenonucleosides, and deazanucleosides, the structures of which are shown in FIGS. 5-11. Those structures in FIGS. 5-11 which are shown surrounded by boxes have been either previously reported or found to be fluorescent during development of the present invention.
Uncharacterized oligomers containing fluorescent analogs were prepared by Ward and colleagues for physical studies using then available nucleoside polymerase enzymes (Ward et al. [1969] J. Biol. Chem. 244:3243-3250; Ward et al. [1969] loc cit 1228-1237). There have been no recent reports in the literature of attempts to combine the use of fluorescent nucleosides or their structural analogs with the synthesis or hybridization techniques of molecular biology or to synthesize fluorescent oligonucleotides therefrom.